The mycobacteria are a group of organisms responsible for several important human diseases. Perhaps the most devastating of these is tuberculosis which kills three million people worldwide and has become dramatically more prevalent in the United States over the past five years. This increase results partly from the increase in HIV infection and has been accompanied by the appearance of drug resistant and multidrug resistant strains of Mycobacterium tuberculosis, the causative agent. Effective control of tuberculosis requires improved and more rapid diagnostic methods, more efficacious vaccines, and better antimycobacterial drugs, particularly for treatment of multidrug resistant infections. While genetic approaches should facilitate these advances, it is only recently that genetic dissection of the mycobacteria has been possible and there is still little known about mycobacterial gene structure, gene expression and its regulation. In addition, there are relatively few tools available for genetic manipulation of mycobacterial cells. Viruses have proven to be powerful tools for genetic analysis of a broad range of organisms, and the viruses of mycobacteria (mycobacteriophages) are no exception. The use of mycobacteriophages was instrumental in the establishment of mycobacterial genetics and the creation of cloning vectors for the introduction of DNA into mycobacteria. More recently, recombinant reporter mycobacteriophages have been proposed as clinical tools for rapid determination of drug susceptibilities of clinical isolates of M. tuberculosis. This project proposes to elucidate the mechanisms of mycobacterial gene expression using mycobacteriophage L5 and its relatives as a model system. L5 is the best characterized for all of the mycobacteriophages and harbors transcriptional promoters that are both powerful and regulated, providing a simple experimental system. These studies will contribute to the construction of sensitive and specific diagnostic reporter phages, tools for construction of recombinant mycobacterial vaccines and systems for analyzing mycobacterial gene expression.